1. Field:
This disclosure relates generally to viral inactivation processes and specifically to viral inactivation in biologically active, therapeutic proteinaceous products.
2. Prior Art:
The safety of pharmaceutical products is always a concern, especially in cases where viral contamination is possible (e.g. in products derived from blood, or cell culture systems designed to produce biologically active proteins). Unfortunately, the very products in which viruses are found are commonly labile and quite sensitive to many known and conventional viral inactivation techniques. Also, in some cases, efforts to protect the protein also protect the virus.
Various attempts have been made to overcome this situation. For example, it is well known that biologically active proteins can be rendered inactive by certain controlled heat treatments or specifically chosen chemical agents. Several heat treatment methods have been devised to inactivate viruses without adversely affecting the biological activity of the protein or significantly reducing its amount. See, for example, U.S. Pat. No. 4,440,679, to Fernandez and Lundblad (carbohydrate stabilizers for pasteurization of the very labile coagulation protein known as Factor VIII) and U.S. Pat. No. 4,762,714 to Mitra and Mozen (showing viral inactivation in an immune globulin product by controlled conditions of pH, temperature and time). See also, U.S. Pat. No. 4,456,590 to Reubenstein showing that Factor VIII can be subjected to pasteurization conditions (at least 60.degree. C. for 10 hours) if first lyophilized, and U.S. Pat. No. 4,495,278 to Thomas (similar heat treatment of lyophilized Factor IX).
Various chemical methods have also been used to inactivate viruses. See, for example, U.S. Pat. No. 4,534,972, to Lembach (use of copper phenanthroline and related compounds) and U.S. Pat. No. 4,481,189 to Horowitz (use of tri-n-butyl phosphate and related compounds).
Carboxylic acids such as caprylic acid have been used in both the preparation of plasma products (precipitation of globulins) and even for the inactivation of lipid-coated virus, but not in the presence of therapeutic biologically active proteins (see J. A. Sands et al., Anti Microbial Agents and Chemotherapy, Jan. 1979, p. 134-136). Carboxylic acids (sodium caprylate) have also been used in combination with heat and amino acids for the viral inactivation of Factor VIII (see U.S. Pat. No. 4,446,134 to Naito et al.). See also, the sequential use of fatty acids for viral inactivation of plasma derivatives as disclosed by Horowitz et al., Vox. Sang. 54:14-20 (1988).
The precipitation of the bulk of the plasma proteins with caprylic acid without affecting IgG, ceruloplasmin and IgA has been described (Steinbuch, M. and Audran, R., Arch. Biochem. Biophys., 134, 279-294 [1969]). Human, equine, ovine and rabbit sera or plasma were diluted with 0.06 M acetate buffer to approximately 1.7% protein, adjusted to pH 4.8 at 20.degree. C., and made 0.174 M (2.5 wt %) with respect to caprylic acid. Attention to buffer molarity (0.06M) and pH (pH 4.8.+-.0.05) were critical to high purity IgG.
The precipitation method of Steinbuch, M., supra, has been applied to spent medium of hybridoma cultures and ascitic fluid from mice, using caprylic acid at a concentration of 0.066 M (0.86 wt %) for recovery of IgG (Russo, C., Callegaro, L., Lanza, E., Ferrone, S., J. Immunol. Methods, 65, 269-271 [1983]). The same method was applied to diluted human plasma adjusted to 0.15 M caprylic acid, or 2.16 wt %, (Habeeb, A.F.S.A. and Francis, E.R., Prep. Biochem., 14(1), 1-17 [1984]).
IgA isolated from Cohn cold ethanol Fraction III by DEAE cellulose adsorption and elution was further purified for removal of alpha-2 macroglobulin by caprylic acid precipitation (Pejaudier, L., Audran, R. and Steinbuch, M., Vox Sang., 23, 165-175 [1972]). Parameters for precipitation consisted of 1.5 to 2.0% protein concentration adjusted to 0.9% sodium chloride, pH 5, and caprylic acid added at room temperature to 0.078 M or 1.12 wt %. The precipitated alpha-2 macroglobulin was removed by centrifugation.
Caprylic acid was used to precipitate most proteins and lipoproteins other than the immunoglobulins present in Cohn cold ethanol Fraction III (Steinbuch, M., Audran, R., Pejaudier, L., Blatrix, C., Prep. Biochem., 3(4), 363-373 [1973]). A suspension of Fraction III at approximately 2.5% protein was adjusted to 0.05 M acetate at pH 4.8 and brought to room temperature. Caprylic acid was added to 0.174 M or 2.5 wt % concentration. The resulting precipitate was discarded. The supernatant was enriched with IgG, IgM and IgA. It should be noted that in all cases where caprylic acid is used as a precipitating agent, it is present in an amount considerably above its maximum solubility in water under ideal conditions of pH, temperature and availability of caprylic acid (as discussed below). Such amounts, commonly about 0.86-2.5 wt %, are needed to assure sufficient quantity of the relatively insoluble caprylic acid in an insoluble form (an emulsion), thus useful as a precipitating agent.
Despite the above numerous publications, we are unaware of any method which uses caprylic acid in less than a precipitating concentration and alone to inactivate substantially all lipid-coated viruses without adversely affecting the biological activity or recoverable amount of therapeutic proteins. We have found that by carefully controlling the conditions of its use, we can use caprylic acid to inactivate lipid enveloped viruses in a biologically active therapeutic product without adversely affecting the activity of the product. Details of our findings are shown below.